Amyotrophic lateral sclerosis (ALS) is a poorly understood neurodegenerative disease with no effective treatment. ALS is connected with military service and veterans are at a higher risk of developing this debilitating disease. Mutations in several genes have been discovered to cause a subset of familial ALS, including copper- zinc superoxide dismutase (SOD1), Fused in Sarcoma (FUS), and TAR DNA-binding protein 43 (TDP-43). Studying familial ALS using the well-defined genetic models will provide critical insights into the disease pathology as well as novel targets for future therapeutic development. This project is focused on the RNA binding protein FUS that has been implicated in both familial and sporadic ALS. ALS-related mutations in FUS cause a liquid-liquid phase separation (LLPS) of the FUS protein, forming liquid droplets in vitro. In neurons, mutant FUS accumulates in the cytoplasm, forming ribonucleoprotein granules and inclusions, eventually leading to neurotoxicity. We recently identified cellular proteins in FUS- positive inclusions and a bioinformatics analysis revealed two previously unknown pathways affected by mutant FUS: protein translation and mRNA surveillance. We also demonstrated that ALS FUS mutations indeed suppressed protein translation and hyper-activated the nonsense-mediated decay (NMD) of mRNAs. Our overarching hypothesis is that the dysregulation of protein translation and mRNA nonsense-mediated decay contributes to FUS toxicity and motor neuron dysfunction. Since little is known about how the mutant FUS- induced dysregulation of protein translation and NMD cause toxicity in neurons, discoveries in this project will fill this knowledge gap and advance the field significantly. Three specific aims are designed to test the hypothesis. Aim 1 is to determine what properties of mutant FUS drive the dysregulation of protein translation and NMD. We will use different domain truncations of FUS to manipulate LLPS and examine the relationship between LLPS and mutant FUS dysregulation. In addition, we will manipulate RNA binding of FUS and test whether RNA binding is a significant contributor to mutant FUS dysfunction. We will also use FUS knockout models to examine whether FUS plays a role in in protein translation and NMD under physiological conditions. Aim 2 is to determine whether mutant FUS impairs translation of specific proteins and NMD of specific mRNAs. We will employ specialized proteomic and transcriptomic approaches to identify changes of nascent protein biosynthesis and mRNA turnover rate as a consequence of mutant FUS. Differentially altered proteins and mRNAs will be validated in animal models as well as iPSC and induced motor neurons derived from familial ALS patients. Results from these -omics approaches will be integrated to determine whether mutant FUS impairs specific molecular and cellular function and pathways. Aim 3 is to examine whether attenuation of NMD hyperactivity can restore protein translation and mitigate FUS neurotoxicity. We will use genetic and pharmacological approaches to attenuate NMD hyperactivity and determine whether the intervention can restore the balance and mitigate FUS toxicity in vitro and in vivo models. The proposed studies are highly innovative, both conceptually and technically. The proposal is based on a number of novel observations regarding the impact of ALS mutant FUS on protein translation and mRNA decay. The results from this project are expected to produce new mechanistic insights into FUS ALS, laying a foundation for future therapeutic development of new ALS treatment(s).